Therapy Resistance In Glioblastoma Research Articles

EPCO-37. LONGITUDINAL TUMOR-WIDE SAMPLING OF GLIOBLASTOMA REVEALS EARLY EVOLUTIONARY DIVERGENCE OF RECURRENCE AND CLUES TOWARDS IDENTIFYING THE BIOLOGICAL TUMOR CENTER

Abstract Therapy resistance in glioblastoma is in part attributed to intratumoral heterogeneity and treatment-resistant cell populations. Understanding heterogeneity requires more complete tumor sampling at diagnosis and at recurrence. We used a whole-tumor sampling approach to obtain 43 spatially mapped biopsies from 3 glioblastomas at diagnosis and recurrence, Pyclone and ClonEvol imputed clonal evolution from exome data, and weighted gene co-expression network analysis inferred gene expression programs from RNA sequencing. Across the cohort, tumor-wide clonal alterations representing evolutionarily early expansions included canonical changes (i.e. Chr7 gain, EGFR amplification) and a diverse set of copy number variations (Chr19/20 gain), driver mutations (i.e. PTEN, KDR), and fusions (LIMCH1::UCHL1, KANK::DOCK8). In 2/3 patients, the founding clone was the only subclone common to primary and recurrence samples and 37% of cancer drivers across the cohort appeared after evolutionary divergence of the primary and recurrent tumors. We identified thirty-two transcriptional programs, six of which were differentially expressed between timepoints. Programs enriched for mitochondrial activity, endothelium/pericytes and classical glioblastoma subtype signature were more highly expressed in primary samples. Programs enriched for mesenchymal-like state, oligodendrocytes and inhibitory interneurons were more highly expressed in recurrence samples. At diagnosis, 11/16 samples showed higher expression of the classical subtype program compared to mesenchymal-like, but at recurrence the mesenchymal program dominated in 18/18 samples. Expression of the programs correlated more strongly with position relative to the contrast-enhancing rather than T2 tumor centroid (p<0.001). These findings reveal novel diversity and complexity in the genetic roots of individual glioblastoma. Recurrences arose from subclones diverging early in evolution of the primary tumor and contained clonal drivers not detected in the primary. Expression data revealed transition from an intratumorally heterogeneous mixture of classical and mesenchymal signatures to tumor-wide mesenchymal at recurrence and suggests that the contrast enhancing centroid may serve as the biological center of the tumor.

TMIC-42. CONQUERING SPP1+ MYELOID-DRIVEN RESISTANCE OF GLIOBLASTOMA TO IL13RA2-TARGETED CAR T CELL THERAPY

Abstract In the evolving landscape of glioblastoma (GBM) therapy, the recent proliferation of clinical trials exploring chimeric antigen receptor (CAR) T cell interventions has garnered attention, although their therapeutic efficacy remains limited. Despite recent progress in elucidating the role of the GBM microenvironment in immunotherapy resistance, particularly regarding intercellular networks and macrophages, significant ambiguities endure, impeding a comprehensive understanding of resistance mechanisms amidst the patient-specific heterogeneity of the tumor microenvironment (TME). In this study, we utilized single-cell RNA sequencing to analyze tumors from 41 glioma patients undergoing IL13Rα2-targeted CAR T cell therapy. Our findings unveiled heightened suppressive extracellular network signatures, particularly SPP-1, predominantly observed within myeloid cells from non-responsive patients. The clinical significance of both SPP1+ myeloid cell abundance and overall expression levels revealed associations with patient outcomes. Subsequently, transcriptomic insights were translated to myeloid cell functions in patients exhibiting high versus low SPP1 expression. SPP1-expressing macrophages displayed diminished antigen presentation, phagocytosis, and cell-mediated cytotoxicity, along with augmented extracellular matrix biogenesis and degradation, iron efflux, and suppressive interleukin pathways. Moreover, SPP1-high macrophages exhibited enhanced ligand-receptor interactions, implying a predisposition towards suppressive activities. Immunometabolism pathways, including lipid and ketone body biogenesis and regeneration, were upregulated in SPP1-expressing macrophages, indicating an increased reliance on non-glycolysis-mediated ATP production. To assess translational implications, the therapeutic efficacy of SPP1 blockade was evaluated in a syngeneic glioma model. Blockade of SPP1 with an anti-SPP1 antibody prior to CAR T cell infusion effectively mitigated the immunosuppressive milieu, reversing resistance in preclinical GBM models to murine IL13Rα2-targeted CAR T cell therapy. Subsequent profiling of tumors post-treatment provided insights into TME alterations following SPP1 blockade therapy. This investigation illuminates the intricate interplay between the TME and CAR T cell therapy resistance in GBM, underscoring SPP1 as a promising therapeutic target to surmount CAR T cell resistance and enhance treatment outcomes.

CNSC-57. THE ROLE OF NEURON-TO-GLIOMA SYNAPTIC COMMUNICATION IN THERAPEUTIC RESISTANCE

Abstract Glioblastomas, primary brain tumors known for their extensive brain colonization and high therapeutic resistance, pose significant treatment challenges. Recent research has revealed that glioblastoma cells can form functional multicellular networks both with themselves, surrounding astrocytes and neurons. However, the investigation of the glioblastoma neuronal connectome has been limited by a lack of suitable methodologies. In this study, we utilized an adapted rabies-based retrograde tracing approach to characterize tumor-connected neurons and examine the neuronal connectome of the tumor in the context of therapy resistance. Our initial findings demonstrated that radiotherapy induces hyperexcitability in tumor-connected neurons, as shown by patch-clamp electrophysiology. This hyperexcitability was associated with an increase in neuron-tumor connectivity post-irradiation. To mitigate this increased neuronal input to therapy-resistant glioblastoma cells, we combined radiotherapy with the anti-epileptic drug perampanel. This combination therapy, which inhibits overall neuronal activity while delivering tumor cell-toxic photon irradiation, proved significantly more effective in reducing the number of therapy-resistant tumor cells. In summary, we employed a novel methodology to investigate the role of the neuronal tumor connectome in therapeutic resistance, identifying tumor-connected neurons as a potential target for improving glioblastoma treatment.

Dual targeting macrophages and microglia is a therapeutic vulnerability in models of PTEN-deficient glioblastoma.

Tumor-associated macrophages and microglia (TAMs) are critical for tumor progression and therapy resistance in glioblastoma (GBM), a type of incurable brain cancer. We previously identified lysyl oxidase (LOX) and olfactomedin like-3 (OLFML3) as essential macrophage and microglia chemokines, respectively, in GBM. Here, single-cell transcriptomics and multiplex sequential immunofluorescence followed by functional studies demonstrate that macrophages negatively correlate with microglia in the GBM tumor microenvironment. LOX inhibition in PTEN-deficient GBM cells upregulates OLFML3 expression via the NF-κB-PATZ1 signaling pathway, inducing a compensatory increase of microglia infiltration. Dual targeting macrophages and microglia via inhibition of LOX and the CLOCK-OLFML3 axis generates potent anti-tumor effects and offers a complete tumor regression in more than 60% of animals when combined with anti-PD1 therapy in PTEN-deficient GBM mouse models. Thus, our findings provide a translational triple therapeutic strategy for this lethal disease.

Metabolic profiling of glioblastoma stem cells reveals pyruvate carboxylase as a critical survival factor and potential therapeutic target.

Glioblastoma (GBM) is a highly aggressive tumor with unmet therapeutic needs, which can be explained by extensive intra-tumoral heterogeneity and plasticity. In this study, we aimed to investigate the specific metabolic features of Glioblastoma stem cells (GSC), a rare tumor subpopulation involved in tumor growth and therapy resistance. We conducted comprehensive analyses of primary patient-derived GBM cultures and GSC-enriched cultures of human GBM cell lines using state-of-the-art molecular, metabolic, and phenotypic studies. We showed that GSC-enriched cultures display distinct glycolytic profiles compared with differentiated tumor cells. Further analysis revealed that GSC relies on pyruvate carboxylase (PC) activity for survival and self-renewal capacity. Interestingly, inhibition of PC led to GSC death, particularly when the glutamine pool was low, and increased differentiation. Finally, while GSC displayed resistance to the chemotherapy drug etoposide, genetic or pharmacological inhibition of PC restored etoposide sensitivity in GSC, both in vitro and in orthotopic murine models. Our findings demonstrate the critical role of PC in GSC metabolism, survival, and escape to etoposide. They also highlight PC as a therapeutic target to overcome therapy resistance in GBM.

Longitudinal tumor-wide sampling of glioblastoma reveals diverse genomic drivers of the earliest clonal expansion at diagnosis and recurrence.

2009 Background: Despite decades of clinical trials, glioblastoma (GBM) remains a rapidly fatal disease. Much of therapy resistance in GBM is attributed to intratumoral heterogeneity in which subclones rapidly evolve and treatment-resistant cell populations emerge. Understanding heterogeneity at diagnosis and its relationship to the origins of recurrence are critical to selecting therapies that are efficacious across the entire tumor. However, few genomic studies go beyond analyzing single tumor samples per patient. Methods: A spatially oriented, whole tumor sampling approach was used to obtain 43 biopsies from 3 GBMs at diagnosis and recurrence. Pyclone and ClonEvol were applied to whole exome sequencing to reconstruct clonal evolution, FACETs identified copy number variations and HATCHet estimated tumor purity. Candidate driver mutations were evaluated in relation to the Catalogue of Somatic Mutations In Cancer. Results: A single founding clone and multiple subclones were identified for each diagnosis-recurrence pair. Tumor-wide clonal alterations representing initial clonal expansions of these GBMs included both canonical changes (Chr 7 gain, Chr 10 loss, CDKN2A deletion, EGFR amplification) and a diverse set of large-scale copy number variations (Chr 19, 20 gain), driver mutations (PTEN, KDR, CDH11, CNTNAP2), and fusions (LIMCH1::UCHL1, KANK::DOCK8). A second subset of alterations (Chr 8 gain, ATRX mutation), appeared to be tumor wide at diagnosis but were not identified at recurrence. Cancer drivers were also present subclonally, including CDKN2A deletion, MDM2 amplification, and mutations in NF1, and GRM3. Evolutionary trees consisted of 5 generations of clones in Patient 323 (P323), 3 in P454, and 4 in P534. Divergence of the recurrent tumors from their matched primary occurred in the second generation in P454 and P534 and in the third in P323. As a result, an average of 37% of potential drivers of oncogenesis and clonal expansion across the cohort appear after divergence. Furthermore, each recurrent tumor contained at least one tumor wide driver alteration that was subclonal or undetected at diagnosis. Conclusions: Whole-tumor sampling of three GBM patients at both diagnosis and recurrence identified a diversity of genomic drivers and deeper and more complex genetic roots of individual GBM than previously seen in single-biopsy studies. Tumor recurrences consistently arose from a single subclone that diverged early in evolution of the primary tumor and contained clonal drivers either not detected or subclonal in the primary—suggesting a role for these drivers in persistence and expansion. In the age of personalized medicine, our study highlights the clinical potential of tumor wide sampling in identifying therapeutic targets that could avoid heterogeneity-related therapeutic failures.

Synergistic Effect of a Combination of Proteasome and Ribonucleotide Reductase Inhibitors in a Biochemical Model of the Yeast Saccharomyces cerevisiae and a Glioblastoma Cell Line.

Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors.

CRISPR/Cas9-Mediated Gene Therapy for Glioblastoma: A Scoping Review.

This scoping review examines the use of CRISPR/Cas9 gene editing in glioblastoma (GBM), a predominant and aggressive brain tumor. Categorizing gene targets into distinct groups, this review explores their roles in cell cycle regulation, microenvironmental dynamics, interphase processes, and therapy resistance reduction. The complexity of CRISPR-Cas9 applications in GBM research is highlighted, providing unique insights into apoptosis, cell proliferation, and immune responses within the tumor microenvironment. The studies challenge conventional perspectives on specific genes, emphasizing the potential therapeutic implications of manipulating key molecular players in cell cycle dynamics. Exploring CRISPR/Cas9 gene therapy in GBMs yields significant insights into the regulation of cellular processes, spanning cell interphase, renewal, and migration. Researchers, by precisely targeting specific genes, uncover the molecular orchestration governing cell proliferation, growth, and differentiation during critical phases of the cell cycle. The findings underscore the potential of CRISPR/Cas9 technology in unraveling the complex dynamics of the GBM microenvironment, offering promising avenues for targeted therapies to curb GBM growth. This review also outlines studies addressing therapy resistance in GBM, employing CRISPR/Cas9 to target genes associated with chemotherapy resistance, showcasing its transformative potential in effective GBM treatments.

Abstract A120: Differentilal roles of PI3K catalytic kinases in glioblastoma's chemoresistance

Abstract Resistance to chemotherapies such as temozolomide presents a major hurdle to effectively treat glioblastoma. This challenge arises possibly from the activation of phosphatidylinositol 3-kinase (PI3K) signaling, which makes PI3K an appealing therapeutic target. However, non-selectively blocking the four highly homologous PI3K catalytic kinases p110α, β, δ, and γ has yielded undesired clinical outcomes. It is therefore imperative to investigate individual PI3K kinases in glioblastoma’s chemosensitivity. Here we report that PI3K kinases differentially regulate chemosensitivity. We found that the four kinases were unequally expressed in grade II/III glioma and glioblastoma with levels of p110β being the highest. Concomitantly, glioblastoma patients deficient of O6-methylguanine-DNA-mehtyltransferase and expressing high levels of p110β and low levels of other kinases (p110β-high) were more likely resistant to temozolomide, thereby experiencing poor prognosis. Perturbation of p110β, but not other kinases, sensitized p110β-high glioblastoma cells or mouse xenograft tumors to temozolomide. Moreover, p110β-selective inhibitors and temozolomide synergistically mitigated the growth of resilient glioblastoma stem cells. Our results have demonstrated the essential role of p110β in regulating chemosensitivity, suggesting that selectively blocking p110β is an effective chemosensitizer for therapy-resistant glioblastoma. Citation Format: Zhi Sheng. Differentilal roles of PI3K catalytic kinases in glioblastoma's chemoresistance [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A120.

TMIC-19. TARGETING TUMOR-MYELOID CELL SYMBIOSIS IN GLIOBLASTOMA

Abstract Heterotypic interactions across diverse cell types of the tumor microenvironment can enable tumor progression and offer points for potential therapeutic intervention. Employing glioblastoma (GBM), a lethal form of brain tumor in adults, as a model system, we sought to (i) understand the nature of specific oncogenic signals in glioma cells that affects myeloid cell biology; (ii) identify myeloid cell factors which may support glioma growth and survival, (iii) develop novel therapeutic strategies targeting the tumor-myeloid cell symbiosis; and (iv) investigate whether blockade of this symbiosis can overcome the primary resistance of immunotherapy. Specifically, the novel oncogenic signals (e.g., CLOCK and TFPI2) and myeloid cell components of microglia and myeloid-derived suppressor cells will be discussed and highlighted. The findings from our studies demonstrate that the newly identified context-dependent tumor-myeloid cell symbiotic interactions not only promote tumor progression but also induce immune checkpoint inhibitor (ICI) therapy resistance in GBM. Thus, our studies provide novel therapeutic targets and offer a great potential to overcome immunotherapy resistance in GBM.

PREDICTING GLIOBLASTOMA GENE EXPRESSION THERAPY RESPONSE WITH MACHINE LEARNING

Abstract AIMS The rapid recurrence of therapy-resistant glioblastoma leaves patients with a dismal 2-year survival rate of just 16%, highlighting the need to develop treatments that combat or prevent glioblastoma recurrence. Recently, glioblastoma patients have been stratified by their gene expression response to therapy. Specifically, genes with JARID2 binding sites in their promoter (JBS-genes) either become up- or down-regulated on progression from the primary to the recurrent tumour. Importantly, these two responses appear to employ different mechanisms of therapy resistance. Using gene expression data from primary tumours, we aim to predict the direction of JBS- gene dysregulation. Ultimately, this prediction could guide treatment decisions that prevent the acquisition of therapy resistance in glioblastoma. METHOD We used gene expression data from 84 primary glioblastoma tumours that had been labelled as either ‘Up’ or ‘Down’ responders based on the direction of JBS-gene dysregulation in matched recurrent tumours. We converted the RNA-sequencing data from the primary tumours to single-sample pathway scores, which were used to train several binary classification machine learning models. RESULTS A ridge regression model achieved an accuracy of 73.9% in predicting the direction of JBS-gene dysregulation in an independent set of 23 primary glioblastoma tumours. CONCLUSIONS Our classifier performs better than random chance demonstrating that gene expression data is informative and molecular features influence this gene expression response to therapy. However, there remains scope for improvement. This suggests further studies may benefit from integrating multiple data types such as genomics, epigenomics, tumour architecture, or clinical variables.

A Syx-RhoA-Dia1 signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in glioblastoma.

Glioblastomas (GBM) are aggressive tumors that lack effective treatments. Here, we show that the Rho family guanine nucleotide exchange factor Syx promotes GBM cell growth both in vitro and in orthotopic xenografts derived from patients with GBM. Growth defects upon Syx depletion are attributed to prolonged mitosis, increased DNA damage, G2/M cell cycle arrest, and cell apoptosis, mediated by altered mRNA and protein expression of various cell cycle regulators. These effects are phenocopied by depletion of the Rho downstream effector Dia1 and are due, at least in part, to increased phosphorylation, cytoplasmic retention, and reduced activity of the YAP/TAZ transcriptional coactivators. Furthermore, targeting Syx signaling cooperates with radiation treatment and temozolomide (TMZ) to decrease viability in GBM cells, irrespective of their inherent response to TMZ. The data indicate that a Syx-RhoA-Dia1-YAP/TAZ signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in GBM and argue for its targeting for cancer treatment.

BLM helicase overexpressed in human gliomas contributes to diverse responses of human glioma cells to chemotherapy

Most of anti-tumour therapies eliminate neoplastic cells by introducing DNA damage which ultimately triggers cell death. These effects are counteracted by activated DNA repair pathways to sustain tumour proliferation capacity. RECQL helicases family, including BLM, participate in DNA damage and repair, and prevent the replication stress. Glioblastoma (GBM) is a common, malignant brain tumour that inevitably recurs despite surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Expression and functions of the BLM helicase in GBM therapy resistance have not been elucidated. We analysed expression and localisation of BLM in human gliomas and several glioma cell lines using TCGA datasets, immunostaining and Western blotting. BLM depleted human glioma cells were generated with CRISPR/Cas9 system. Effects of chemotherapeutics on cell proliferation, DNA damage and apoptosis were determined with flow cytometry, immunofluorescence, Western blotting and RNA sequencing. We found upregulated BLM mRNA levels in malignant gliomas, increased cytosolic localisation and poor survival of GBM patients with high BLM expression. BLM deficiency in LN18 and LN229 glioma cells resulted in profound transcriptomic alterations, reduced cell proliferation, and altered cell responses to chemotherapeutics. BLM-deficient glioma cells were resistant to the TMZ and PARP inhibitor treatment and underwent polyploidy or senescence depending on the TP53 activity. Our findings of high BLM expression in GBMs and its roles in responses to chemotherapeutics provide a rationale for targeting BLM helicase in brain tumours. BLM deficiency affects responses of glioma cells to chemotherapeutics targeting PARP1 dependent pathways.

Development of a Synthetic, Injectable Hydrogel to Capture Residual Glioblastoma and Glioblastoma Stem-Like Cells with CXCL12-Mediated Chemotaxis.

Glioblastoma (GBM), characterized by high infiltrative capacity, is the most common and deadly type of primary brain tumor in adults. GBM cells, including therapy-resistant glioblastoma stem-like cells (GSCs), invade the healthy brain parenchyma to form secondary tumors even after patients undergo surgical resection and chemoradiotherapy. New techniques are therefore urgently needed to eradicate these residual tumor cells. A thiol-Michael addition injectable hydrogel for compatibility with GBM therapy is previously characterized and optimized. This study aims to develop the hydrogel further to capture GBM/GSCs through CXCL12-mediated chemotaxis. The release kinetics of hydrogel payloads are investigated, migration and invasion assays in response to chemoattractants are performed, and the GBM-hydrogel interactions in vitro are studied. With a novel dual-layer hydrogel platform, it is demonstrated that CXCL12 released from the synthetic hydrogel can induce the migration of U251 GBM cells and GSCs from the extracellular matrix microenvironment and promote invasion into the synthetic hydrogel via amoeboid migration. The survival of GBM cells entrapped deep into the synthetic hydrogel is limited, while live cells near the surface reinforce the hydrogel through fibronectin deposition. This synthetic hydrogel, therefore, demonstrates a promising method to attract and capture migratory GBM cells and GSCs responsive to CXCL12 chemotaxis.

Abstract 1665: Imipramine enhances the efficacy of lomustine by regulating WNT-β-catenin signaling in glioblastoma

Abstract Background: Glioblastoma (GBM) is one of the most aggressive and difficult-to-treat brain cancers. Surgical excision followed by concurrent temozolomide (TMZ), and radiation therapy are the current standard of care for GBM. Lomustine, another alkylating regimen, has also been approved for the treatment of patients with recurrent GBM in addition to TMZ. However, the primary GBM frequently recur as therapy-resistant relapses resulting in shorter survival of patients. To date, no systemic therapies have shown proven survival benefit in recurrent GBM. In this study, we examined whether FDA approved anti-depressant drug Imipramine serve as the potent option for treating patients with recurrent GBM and to improve efficacy of approved chemotherapy. Methods: We investigated the anticancer effects of Imipramine using both established and patient-derived GBM cell lines. We tested the effect of Imipramine and Lomustine combination therapy using cell viability, colony formation, migration and apoptosis assays. Mechanistic studies were conducted using RNA-seq, western blotting, RT-PCR and reporter assays. In vivo efficacy of combination therapy was evaluated using orthotopic mouse models. Results: Our results showed that antidepressant Imipramine blocked the growth of recurrent GBM. Furthermore, Imipramine enhanced the response of Lomustine in therapy resistant GBM cells as a combination therapy and significantly reduced the cell survival and migration, and increased the apoptosis in both established and patient derived GBM cells. Further, our results demonstrated that Imipramine inhibits growth of self-renewing cancer stem cells, which contribute to drug resistance and relapse of GBM. Mechanistic studies revealed that the Imipramine treatment abrogated the WNT-β-catenin signaling, which was confirmed by TOPFLASH reporter assay. Conclusion: Collectively, the results from our study suggest that Imipramine alone and in combination with Lomustine can be used for treating recurrent GBM. Based on these results, a clinical trial is currently ongoing, where we are testing the efficacy of Imipramine for treating patients with recurrent GBM. Citation Format: Prabhakar Pitta Venkata, Arhan Rao, Santosh Timilsina, Panneerdoss Subbarayalu, Andrew Brenner, Gangadhara Reddy Sareddy, Manjeet Rao, William Kelly, Ratna Vadlamudi. Imipramine enhances the efficacy of lomustine by regulating WNT-β-catenin signaling in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1665.

CSIG-04. BMP4 INDUCTION OF A SENESCENCE-LIKE PHENOTYPE IN HUMAN GLIOBLASTOMA CELLS IS PARTIALLY DEPENDENT ON P21

Abstract Bone morphogenetic protein 4 (BMP4) was initially suggested as a potential differentiation-inducing factor to be used in the therapy of glioblastoma. We and others have however demonstrated that the response is reversible, variable among patient samples, and heterogeneous within the same cell line. To deepen our knowledge on how BMP4 affects different types of glioblastoma cells, we treated phenotypically different clones from the same patient tumor—a multitherapy-sensitive/proneural-like (SENS/PN) clone and a multitherapy-resistant/mesenchymal-like (RES/MES) clone—with BMP4. In the SENS/PN clone, BMP4 turned on a mesenchymal-related gene program, whereas this response was less prominent in the RES/MES clone. Untreated SENS/PN cells were smaller than RES/MES cells, but both clones responded to BMP4 by cell size enlargement. Increase in cell size has been suggested to precede senescence; young cells are smaller than old cells that eventually enter replicative senescence. BMP4 induced a senescence-like phenotype in a subpopulation of cells, demonstrated by induction of senescence-associated (SA)-β-gal, p21 up-regulation, lamin B1 down-regulation, as well as increased lysosomal mass and granularity. This was more pronounced in the RES/MES clone than in the SENS/PN clone, and it was dependent on canonical SMAD signaling. Senolytic treatment ablated the SA-β-gal positive cells and reduced the p21 level at the population level. Targeted deletion of p21 abolished BMP4-induced SA-β-gal and increase in cell growth, while lamin B1 down-regulation remained, demonstrating that p21 signaling is crucial for one part of the senescence induction by BMP4. We are currently further investigating a connection between cell size, mesenchymality and senescence. We hypothesize that large mesenchymal-like cells are closer to senescence than smaller proneural cells within the cell culture. A combination of senescence-induction and senolytic treatment may open treatment opportunities to target therapy-resistant glioblastoma cells.

Roles of Chromatin Remodelling and Molecular Heterogeneity in Therapy Resistance in Glioblastoma.

Simple SummaryWe review the role of chromatin and epigenetic dysregulation in therapy resistance in glioblastoma. We discuss how epigenetic and genetic forces may cooperate to programme functional cell states that are inherently resistant to therapy. Targeting epigenetic factors that are dysregulated in this malignancy could, therefore, improve clinical outcomes for patients. We highlight some preclinical and clinical compounds that were tested or are currently being explored for glioblastoma. Lastly, we present our thoughts on the requirements for the development of next-generation epigenetic therapies.Cancer stem cells (CSCs) represent a therapy-resistant reservoir in glioblastoma (GBM). It is now becoming clear that epigenetic and chromatin remodelling programs link the stemlike behaviour of CSCs to their treatment resistance. New evidence indicates that the epigenome of GBM cells is shaped by intrinsic and extrinsic factors, including their genetic makeup, their interactions and communication with other neoplastic and non-neoplastic cells, including immune cells, and their metabolic niche. In this review, we explore how all these factors contribute to epigenomic heterogeneity in a tumour and the selection of therapy-resistant cells. Lastly, we discuss current and emerging experimental platforms aimed at precisely understanding the epigenetic mechanisms of therapy resistance that ultimately lead to tumour relapse. Given the growing arsenal of drugs that target epigenetic enzymes, our review addresses promising preclinical and clinical applications of epidrugs to treat GBM, and possible mechanisms of resistance that need to be overcome.

MEX3A Impairs DNA Mismatch Repair Signaling and Mediates Acquired Temozolomide Resistance in Glioblastoma.

A MEX3A/CCR4-NOT/MSH2 axis plays a crucial role in promoting temozolomide resistance, providing new insights into the function of MEX3A and suggesting MEX3A as a potential therapeutic target in therapy-resistant glioblastoma.

P02.07.B Patient-derived glioblastoma organoids: Elucidating the mechanisms of glioblastoma therapeutic resistance in the context of tumor microenvironment

Abstract Background Intratumoral heterogeneity plays an important role in glioblastoma (GB) resistance to standard therapy consisting of irradiation and chemotherapy with temozolomide (TMZ). However, classical in vitro GB models fail to represent the complex cellular composition of tumors in vivo, which hinders relevant examination of GB therapeutic response. To overcome these limitations, we studied the effects of irradiation and TMZ in a novel patient-derived organoid model. Material and Methods We established a patient-derived GB organoid model by a protocol recently published by Jacob et al. Original tumor tissue and tissue-derived organoids were compared by immunofluorescence staining of selected cell type markers and qPCR analysis of expression levels of a panel of selected target genes, including 15 genes defining GB subtypes. To analyze GB therapeutic response, organoids from 11 patients were exposed to a single dose of irradiation (10 Gy), one-week treatment with TMZ (50 µM) or their combination. The effects of therapy were assessed by viability and invasion assays. Expression levels of a number of genes related to GB subtypes, epithelial-mesenchymal transition, stemness, DNA damage responses, cell cycle, cytokines, and cell markers of the tumor microenvironment (TME) were compared between treated organoids and untreated controls. In addition, the heterogeneity of the TME and its responses to treatment were investigated by spatially resolved transcriptomics with in situ sequencing (ISS) methodology. Results Organoids recapitulate inter-patient variability and reflect the cellular composition and gene expression levels of the tumor tissue from which they were derived. GB stem cells and differentiated cancer cells are present in organoids along with various cells of the TME, e.g., macrophages and microglia, lymphocytes, and endothelial cells. Irradiation and TMZ showed no significant effects on organoid viability and invasion. However, some target genes were differentially expressed in the treated organoids, such as E3 ubiquitin-protein ligase MDM2 and cyclin-dependent kinase inhibitor 1A (CDKN1A). To our knowledge, we are the first to apply spatially resolved transcriptomics (ISS) to formalin-fixed, paraffin-embedded sections of (un)treated GB organoids. Our results elucidate the role of the TME in GB therapeutic response and shed light on potential mechanism underlying GB therapy resistance. Conclusion Patient-derived GB organoids recapitulate the key characteristics and complex composition of patient’s tumor tissue, providing a valuable platform for studies of GB therapeutic response and resistance.

The Oncolytic Adenovirus XVir-N-31, in Combination with the Blockade of the PD-1/PD-L1 Axis, Conveys Abscopal Effects in a Humanized Glioblastoma Mouse Model.

Glioblastoma (GBM) is an obligatory lethal brain tumor with a median survival, even with the best standard of care therapy, of less than 20 months. In light of this fact, the evaluation of new GBM treatment approaches such as oncolytic virotherapy (OVT) is urgently needed. Based on our preliminary preclinical data, the YB-1 dependent oncolytic adenovirus (OAV) XVir-N-31 represents a promising therapeutic agent to treat, in particular, therapy resistant GBM. Preclinical studies have shown that XVir-N-31 prolonged the survival of GBM bearing mice. Now using an immunohumanized mouse model, we examined the immunostimulatory effects of XVir-N-31 in comparison to the wildtype adenovirus (Ad-WT). Additionally, we combined OVT with the inhibition of immune checkpoint proteins by using XVir-N-31 in combination with nivolumab, or by using a derivate of XVir-N-31 that expresses a PD-L1 neutralizing antibody. Although in vitro cell killing was higher for Ad-WT, XVir-N-31 induced a much stronger immunogenic cell death that was further elevated by blocking PD-1 or PD-L1. In vivo, an intratumoral injection of XVir-N-31 increased tumor infiltrating lymphocytes (TILs) and NK cells significantly more than Ad-WT not only in the virus-injected tumors, but also in the untreated tumors growing in the contralateral hemisphere. This suggests that for an effective treatment of GBM, immune activating properties by OAVs seem to be of greater importance than their oncolytic capacity. Furthermore, the addition of immune checkpoint inhibition (ICI) to OVT further induced lymphocyte infiltration. Consequently, a significant reduction in contralateral non-virus-injected tumors was only visible if OVT was combined with ICI. This strongly indicates that for an effective eradication of GBM cells that cannot be directly targeted by an intratumoral OV injection, additional ICI therapy is required.